The enumeration of absolute levels of cells and their subsets in body fluids is of primary importance in determining the state of health of human beings and mammals in general. The primary analytical platform for performing such analyses is flow cytometry in which the specimen is either injected directly or after prior enrichment in rare cell analysis. Flow cytometry and similar complex analytical systems remain largely inaccessible for routine clinical use in resource-poor countries due to high instrument and reagents costs, lack of technical support, lack of robustness requiring frequent service, and the need for AC power. There is a clear need for simpler, more compact and less expensive systems also operable with emergency DC battery power and preferably exhibiting comparable performance characteristics.
In addition to the above-cited full sized flow cytometry systems available for example from Becton Dickinson and Beckman-Coulter, these vendors also sell scaled down less expensive versions, which still suffer from the other cited limitations. Similar limitations apply to the compact CyFlow® from Partec GmbH, (Munster, Germany) and to the Guava Personal Cytometer (Burlingame, Calif.). U.S. Pat. No. 6,097,485 (assigned to Integrated Wave Guides, Brookings, S. Dak.) discloses an ultra-miniature personal flow cytometer (pFCM) claimed to be of lower cost, but still exhibiting rather complex electronic circuitry, optical designs, data reduction, all of which contribute to unacceptable complexity for a third world setting. All these systems use the flow concept, which obviously complicates the instrumental design. These scaled down versions of flow cytometry systems do not meet the clear need for a truly simple, compact, rugged, battery-operable and affordable cell analyzer.
Among the numerous clinical applications for a simple cell analyzer are: counting of CD4 T-lymphocytes in HIV, B-lymphocytes in Chronic Lymphocytic Leukemia, CD34 progenitor cells, granulocytes and platelets in patients treated with chemotherapy, and leukocytes in blood bags. The current systems and methods for cell analysis have some significant disadvantages. They generally require sophisticated techniques, which involve the use of instruments that are expensive both in terms of initial cost and maintenance as well as requiring highly trained personnel. This makes the conventional systems unsuitable for use in laboratories of resource-poor countries or physicians' offices. Therefore, a low-cost, easy-to-use method for cell enumeration such as CD4 enumeration is needed. Such a method may serve as a compact alternative to the current cell analysis systems that would be suitable for physician practices, bedside testing, or in open field settings with the ability to count rare cells in each condition. Further enumerating white cells in, for example, blood bags by a rapid, inexpensive means, instead of using flow cytometry where the analysis time is very long.
The invention described herein meets the criteria above. The system does not require extensive sample preparation. Preparation is achieved by adding a sample through capillary flow to a chip containing reagents in a dried hydrogel layer. A large-area image cytometer with three light sources for the excitation of different fluorochromes and a CCD camera is used as the imaging platform. The prior art contains computer-assisted microscopes. U.S. Pat. No. 5,018,209 teaches a computer driven microscope in which the user manually selects positive events while looking at an image. Obviously, this does not have a high enough throughput to be an effective analyzer, especially in remote settings.
In U.S. Pat. No. 5,287,272, an automated cytological specimen classification system and method is described. This system relies on a complicated neural network to process images of cells based on morphology. While very effective for classifying objects in images, it requires a large amount of computational resources. Furthermore, human input and subsequent analysis is still necessary. Other devices, such as those described in U.S. Pat. Nos. 5,073,857 and 5,077,806, use window sub-image pixel counting algorithms for image analysis by using predetermined thresholds.
Another set of instruments in the prior art is designed as bench top analyzers. In U.S. Pat. No. 5,073,857, pap smears are analyzed by a computer controlled microscope and camera and computer driven image analysis. In U.S. Pat. No. 6,221,607, an automated microscope is described for analyzing in situ hybridization events in a biological specimen.
The devices in the aforementioned prior art are designed to image slides. None are capable of detecting and enumerating a target population within a biological specimen as defined herein. Furthermore, none appear to be portable or high throughput devices. These instruments are designed to rely on a desktop computer to control the microscope and camera, and to perform image analysis. The present invention overcomes many of the difficulties that lie in the prior art.
In US Patent Applications 20100179068 & 20090079963 Clondiag GmbH/Alere GmbH, Jena, (Germany) disclose an instrument and method for point-of-care CD4 counting. Other than the on-chip sample preparation method we disclose here, their test requires active mixing since the reagents are contained in a pellet inside the chamber and therefore do not mix with the sample by diffusion on a useful time scale. The image area is only 1727 μm×1385 μm and the height of the chamber is 0.04 mm, i.e. the volume of sample per image is less than 0.1 μl. Since this is not sufficient for many applications, images of several distinct portions of the sample have to be taken. This can be realized by moving the image area (e.g. by moving the camera) or by moving the sample, either by displacing the whole chamber or by pumping the sample through the chamber. Any of these solutions will increase the complexity of the system compared with the invention we disclose here.
William R. Rodriguez et al. developed another approach for CD4 enumeration based on fluorescence imaging, which has been further developed and produced by LabNow (PLoS Med. 2005 July; 2(7): e182.). This approach uses filters in order to separate leukocytes from erythrocytes. Other than in the method we disclose here, the sample preparation has to be done manually and the exact volume of the blood sample has to be known.
Hydrogels have been used for several decades for the controlled release of molecules. In 1959, several patents U.S. Pat. Nos. 2,886,440-2,886,449 by Gen Foods Corp disclosed the use of gelatin to control the release of flavor from chewing gum. From the 1970s, drug release from gels was studied (U.S. Pat. No. 3,796,217; H. P. Merle and P. Speiser, “Preparation and in vitro evaluation of cellulose acetate phthalate coacervate microcapsules”, J. Pharm. Sci. 62, 1444-1448, (1973)). A review article published in 2005 (S. Young et al., “Gelatin as a delivery vehicle for the controlled release of bioactive molecules”, J. Controlled Release 109, Issues 1-3, 5 Dec. 2005, Pages 256-274) lists many applications for the release of molecules from gelatin. None of these applications are related to in vitro diagnostics or microfluidics. An earlier, similar review article neither includes any application comparable to the invention we disclose here: N. B. Graham and M. E. McNeill, “Hydrogels for controlled drug delivery”, Biomaterials 5, 1984, Pages 27-36.
The possibility to control chemical reactions by diffusion of reagents through a gelatin layer has been known for a long time. It is also the principle underlying the developing and fixation processes of photographic material. See for example U.S. Pat. No. 1,846,300. However, to the best of our knowledge, the controlled release of reagent molecules from hydrogels for diagnostics application in a geometrical configuration that avoids the necessity for additional active mixing has not been disclosed previously.
Due to the small dimensions inside microfluidic devices, flow will usually be laminar. Turbulent flow is difficult to realize in microscopic structures. Therefore, ways to realize efficient on-chip mixing have been of interest over many years. Several recent articles review different solutions for this problem (V. Hessel et al., “Micromixers—a review on passive and active mixing principles”, Chem. Eng Sci 60, 2479-2501 (2005), and N.-T. Nguyen and Z. Wu, “Micromixers—a review”, J. of Micromech. and Microeng 15 R1, doi: 10.1088/0960-1317/15/2/R01 (2005)). The possibility to distribute the reagent inside the device and to slow down the release/mixing of reagents in order to ensure homogeneous concentrations throughout the whole sample is not considered in any of the publications.